The objective of these proposed studies is to better understand the morphological and biochemical properties of synthesis, secretion and extracellular deposition of chondroitin sulfate proteoglycan and interrelated matrix macromolecules. The simultaneous immunohistochemical labeling of two products will be used to visualize coordinate biosynthesis and matrix deposition for specific gene products of individual cells while preserving critical cell-cell and cell-matrix relationships. Chondrocytes in culture will provide a well-controlled and readily manipulated living model system for the study of biosynthesis and interactions among matrix constituents, presumably more representative of in vivo conditions than reconstituted cell-free systems. Antibodies specific for cartilage matrix components (type II collagen, core protein of chondroitin sulfate proteoglycan and link protein(s)) and for matrix components characteristic of fibroblasts and precartilage mesenchyme (type I collagen and fibronectin) will be used. Another dimension to the investigation of biosynthesis will be provided by complementary molecular studies. These experiments are designed to elucidate properties of the co- and/or post-translational processing of the recently identified nascent core protein of chondroitin sulfate proteoglycan. Processing experiments will be extended to consider related cartilage matrix constituents such as type II collagen and link. Results obtained from such cell-free molecular studies will be related to information about processing and biosynthetic intermediates obtained from pulse-labeling of chondrocytes in culture. In order to evaluate further the biosynthesis of matrix macromolecules and interrelationships among them, chondrocytes will be grown in culture in the presence of agents which 1) block cartilage differentiation or 2) alter the synthesis or processing of an individual matrix constituent. The use of immunohistochemistry in conjunction with biochemical and molecular analysis should greatly enhance the total picture of biosynthesis and interaction of matrix components and thereby provide 1) a better understanding of the formation and regulation of cartilage matrix and 2) a firm basis for the examination of proteoglycans and their interactions with other matrix components in non-cartilage connective tissues such as vascular or ocular tissues.